Project Summary/Abstract The long-term goal of this work is to understand the virulence and heat shock responses by which bacteria survive environmental changes. Bacteria respond rapidly to changes by regulating gene expression through an active and complex transcriptome. Bacterial genes are often regulated by temperature-induced changes in RNA structure, which are termed `RNA thermometers' (RNATs). RNATs often function by forming a structure that sequesters the Shine-Dalgarno (SD) sequence, thereby preventing ribosome binding. Discovery and verification of RNATs has historically been conducted by computational methods and in vitro studies. A gap in knowledge exists for understanding RNAT folding in vivo, and the dynamic ability of these structures to control gene expression. To fill this gap, global genome-wide in vivo structure probing will be applied using a method recently developed in this laboratory called Structure-seq2. This technique will be used to probe the transcriptome of Bacillus subtilis, a model Gram-positive bacterial species. While pioneering work led to the description of the first RNATs, using in vivo genome-wide techniques the proposed work will identify RNATs throughout the transcriptome, not just those near SD sequences. Using Structure-seq2 the following Aims will be accomlished: (1) Probe the structural landscape of the B. subtilis transcriptome at low and high temperatures to discover RNA thermometers. RNA structure is known to change in response to temperature. This phenomenon will be explored in the B. subtilis transcriptome. As B. subtilis is a soil bacterium, high and low temperatures will be used to replicate the extremes of natural growth in the environment. Testing will begin at low and high temperatures of 23C and 44C to identify RNA structures that change under these different conditions. This work is supported by preliminary data. (2) Classify diverse bacterial RNA thermometers genome-wide at a series of temperatures. No RNA thermometers have been experimentally characterized from B. subtilis, although many are predicted throughout the genome. By conducting Structure-seq2 at a series of growth temperatures, RNATs will be discovered that have different melting temperatures, function at narrow and wide temperature ranges, and have an instantaneous or gradual response. Results from Aim 1 will be used as a guide for regions likely to contain RNATs. (3) Characterize B. subtilis RNAT function. RNA structures that change in response to changing temperature will be tested as thermometers using bgaB reporter assays. Regions of interest will be cloned as bgaB translational fusions to report on gene expression, and transcriptional fusions will be used for in vitro transcription attenuation assays. This work will result in the first classification of RNATs by temperature midpoints and sensitivity, and will reveal novel regulatory strategies that will be identified in other bacterial species, including human pathogens.